Site-directed mutagenesis of the putative active site of human 17 beta-hydroxysteroid dehydrogenase type 1.
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ABSTRACT:
SUBMITTER: Puranen TJ
PROVIDER: S-EPMC1137484 | biostudies-other | 1994 Nov
REPOSITORIES: biostudies-other
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Project description:Hydroxysteroid 17-beta dehydrogenase type 10 (HSD10) deficiency (HSD10 disease) is a rare X-linked neurodegenerative condition caused by abnormalities in the HSD17B10 gene. A total of 10 mutations have been reported in the literature since 2000. Described phenotypes include a severe neonatal or progressive infantile form with hypotonia, choreoathetosis, seizures, cardiomyopathy, neurodegeneration, and death, as well as an attenuated form with variable regression. Here we present the second report of a c.194T>C (p.V65A) mutation in two half-brothers with a clinical phenotype characterized by neurodevelopmental delay, choreoathetosis, visual loss, cardiac findings, and behavioral abnormalities, with regressions now noted in the older sibling. Neither has experienced a metabolic crisis. Both of the siblings had normal tandem mass spectroscopy analysis of their newborn screening samples. The older brother's phenotype may be complicated by the presence of a 3q29 microduplication. Diagnosis requires a high index of suspicion, as the characteristic urine organic acid pattern may escape detection. The exact pathogenic mechanism of disease remains to be elucidated, but may involve the non-dehydrogenase functionalities of the HSD10 protein. Our report highlights clinical features of two patients with the less fulminant phenotype associated with a V65A mutation, compares the reported phenotypes to date, and reviews recent findings regarding the potential pathophysiology of this condition.Summary Sentence Hydroxysteroid 17-beta dehydrogenase type 10 (HSD10) disease (HSD10 disease) is a rare X-linked neurodegenerative condition with a variable clinical phenotype; diagnosis requires a high index of suspicion.
| S-EPMC5355379 | biostudies-literature
Project description:Rat ovarian 20 alpha-hydroxysteroid dehydrogenase plays a pivotal role in leuteolysis and parturition by catalysing the reduction of progesterone to give the progestationally inactive steroid 20 alpha-hydroxyprogesterone. Putative mechanism based inhibitors of this enzyme were synthesized as potential progestational maintaining agents, including the epimeric allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol and the related vinyl ketone 1-(3 beta-hydroxy-5 alpha-androstan-17 beta-yl)-2-propen-1-one. The vinyl ketone inactivates rat ovarian 20 alpha-hydroxysteroid dehydrogenase, semi-purified by poly(L-lysine)-agarose column chromatography, in a rapid time-dependent manner. Analysis of the pseudo-first-order inactivation plots gave a Ki of 2.0 microM for the inhibitor and a t1/2 for the enzyme of 20 s at saturation. These data indicate that the vinyl ketone is a potent and efficient inactivator of the ovarian dehydrogenase. Neither dialysis in the presence or absence of a competing nucleophile nor gel filtration reserves the inactivation, suggesting that a stable covalent bond is formed between the enzyme and steroid ligand. Both substrates (20 alpha-hydroxyprogesterone and NADP+) protect the enzyme from inactivation; moreover, initial velocity measurements in the presence of saturating concentrations of both substrates indicate that the vinyl ketone can behave as a competitive inhibitor, yielding a Ki value identical with that obtained in the inactivation experiments. Our results imply that the vinyl ketone is an active-site directed alkylating agent. By contrast the allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol are neither substrates nor inhibitors of the ovarian enzyme and appear to be excluded from the catalytic site. The rapid inactivation observed with the vinyl ketone suggests that this compound may be useful as a progestational maintaining agent.
| S-EPMC1147478 | biostudies-other
Project description:Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-β hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.
| S-EPMC4563777 | biostudies-literature
Project description:Hydroxysteroid 17-beta-dehydrogenase 13 (HSD17B13) is a hepatic lipid droplet-associated enzyme that is upregulated in patients with non-alcoholic fatty liver disease. Recently, there have been several reports that predicted loss of function variants in HSD17B13 protect against the progression of steatosis to non-alcoholic steatohepatitis with fibrosis and hepatocellular carcinoma. Here we report crystal structures of full length HSD17B13 in complex with its NAD+ cofactor, and with lipid/detergent molecules and small molecule inhibitors from two distinct series in the ligand binding pocket. These structures provide insights into a mechanism for lipid droplet-associated proteins anchoring to membranes as well as a basis for HSD17B13 variants disrupting function. Two series of inhibitors interact with the active site residues and the bound cofactor similarly, yet they occupy different paths leading to the active site. These structures provide ideas for structure-based design of inhibitors that may be used in the treatment of liver disease.
| S-EPMC10449848 | biostudies-literature
Project description:The data presented here are related to the research article entitled "Estradiol-independent modulation of breast cancer transcript profile by 17beta-hydroxysteroid dehydrogenase type 1" (J.A. Aka, E.L. Calvo, S.X. Lin, 2016) [1]. We evaluated the effect of the steroidal enzyme 17?-HSD1 and its product, the estrogenic hormone 17-beta-estradiol (E2), on gene transcription profile of breast cancer cells. RNA interference technique was used to knock down the 17?-HSD1 gene (HSD17B1) in the hormone-dependent breast cancer cell line T47D in steroid-deprived medium. Transfected cells were subsequently treated with E2, and microarray analyses (with three contrasts) were used to investigate (i) the effect of 17?-HSD1 expression on breast cancer cell transcript profile in steroid-deprived condition, (ii) the effect of E2 on breast cancer gene expression and (iii) if E2 affects gene regulation by 17?-HSD1. Functional enrichments of the differentially expressed genes were assessed using Ingenuity Pathway Analysis (IPA). Here, we showed data on 140 genes that are induced or repressed 1.5 time or higher (p < 0.05) in the HSD17B1-silenced and E2-treated T47D cells revealed by microarray analysis, and presented the 14 functional terms found in the cancer and in the cell death and survival categories revealed by the IPA biological function analysis. Data on IPA Canonical Pathway and network analyses is also presented. Further discussion on gene regulation by 17?-HSD1 and E2 is provided in the accompanying publication [1].
| S-EPMC5122694 | biostudies-literature
Project description:BACKGROUND: During human pregnancy, the placental villi produces high amounts of estradiol. This steroid is secreted by the syncytium, which is directly in contact with maternal blood. Estradiol has to cross placental foetal vessels to reach foetal circulation. The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) was detected in placental endothelial cells of foetal vessels inside the villi. This enzyme catalyzes the conversion of estradiol to estrone, and of testosterone to androstenedione. It was proposed that estradiol level into foetal circulation could be regulated by 17beta-HSD2. METHODS: We obtained placentas from 10 to 26 6/7 weeks of pregnancy from women undergoing voluntary termination of pregnancy, term placentas were collected after normal spontaneous vaginal deliveries. We quantified 17beta-HSD2 mRNA levels in mid-gestation and term human placenta by RT-QPCR. We produced a new anti-17beta-HSD2 antibody to study its spatio-temporal expression by immunohistochemistry. We also compared steroid levels (testosterone, estrone and estradiol) and 17beta-HSD2 mRNA and protein levels between term placenta and endometrium. RESULTS: High 17beta-HSD2 mRNA and protein levels were found in both mid-gestation and term placentas. However, we showed that 17beta-HSD2 mRNA levels increase by 2.27 fold between mid-gestation and term. This period coincides with a transitional phase in the development of the villous vasculature. In mid-gestation placenta, high levels of 17beta-HSD2 were found in mesenchymal villi and immature intermediate villi, more precisely in endothelial cells of the stromal channel. At term, high levels of 17beta-HSD2 were found in the numerous sinusoidal capillaries of terminal villi. 17beta-HSD2 mRNA and protein levels in term placentas were respectively 25.4 fold and 30 to 60 fold higher than in the endometrium. Steroid levels were also significantly higher in term placenta than in the endometrium. CONCLUSION: The spatial and temporal expression of 17beta-HSD2 in the placenta during pregnancy and the comparison of 17beta-HSD2 expression and steroid levels between placental villi and endometrium are compatible with a role in the modulation of active and inactive forms of estrogens. Our observations strongly support the hypothesis that 17beta-HSD2 acts as a barrier decreasing estradiol secretion rates in the foetal circulation.
| S-EPMC1947994 | biostudies-literature
Project description:Numerous studies have reported that oestrogens may contribute to the development of non‑small cell lung cancer (NSCLC). Although different steroidogenic enzymes have been detected in the lung, the precise mechanism leading to an exaggerated accumulation of active oestrogens in NSCLC remains unexplained. 17‑β‑Hydroxysteroid dehydrogenase type 2 (HSD17B2) is an enzyme involved in oestrogen and androgen inactivation by converting 17‑β‑oestradiol into oestrone, and testosterone into 4‑androstenedione. Therefore, the enzyme serves an important role in regulation of the intracellular availability of active sex steroids. This study aimed to determine the expression levels of HSD17B2 in lung cancer (LC) and adjacent histopathologically unchanged tissues obtained from 161 patients with NSCLC, and to analyse the association of HSD17B2 with clinicopathological features. For that purpose, reverse transcription‑quantitative PCR, western blotting and immunohistochemistry were conducted. The results revealed that the mRNA and protein expression levels of HSD17B2 were significantly decreased in LC tissues compared with matched controls (P<10‑6). Conversely, strong cytoplasmic staining of HSD17B2 was detected in the unchanged respiratory epithelium and in glandular cells. Notably, a strong association was detected between reduced HSD17B2 expression and advanced tumour stage, grade and size. Furthermore, it was revealed that HSD17B2 may have potential prognostic significance in NSCLC. A log‑rank test revealed the benefit of high HSD17B2 protein expression for the overall survival (OS) of patients (P=0.0017), and multivariate analysis confirmed this finding (hazard ratio=0.21; 95% confidence interval=0.07‑0.63; P=0.0043). Stratified analysis in the Kaplan‑Meier Plotter database indicated that patients with higher HSD17B2 expression presented better OS and post‑progression survival. This beneficial effect was particularly evident in patients with adenocarcinoma and during the early stages of NSCLC. Decreased expression of HSD17B2 appears to be a frequent feature in NSCLC. Retrospective analysis suggests that the HSD17B2 mRNA and protein status might be independent prognostic factors in NSCLC and should be further investigated.
| S-EPMC7170045 | biostudies-literature
Project description:Background and aims17-Beta hydroxysteroid dehydrogenase 13 (HSD17B13) is genetically associated with human nonalcoholic fatty liver disease (NAFLD). Inactivating mutations in HSD17B13 protect humans from NAFLD-associated and alcohol-associated liver injury, fibrosis, cirrhosis, and hepatocellular carcinoma, leading to clinical trials of anti-HSD17B13 therapeutic agents in humans. We aimed to study the in vivo function of HSD17B13 using a mouse model.Approach and resultsSingle-cell RNA-sequencing and quantitative RT-PCR data revealed that hepatocytes are the main HSD17B13-expressing cells in mice and humans. We compared Hsd17b13 whole-body knockout (KO) mice and wild-type (WT) littermate controls fed regular chow (RC), a high-fat diet (HFD), a Western diet (WD), or the National Institute on Alcohol Abuse and Alcoholism model of alcohol exposure. HFD and WD induced significant weight gain, hepatic steatosis, and inflammation. However, there was no difference between genotypes with regard to body weight, liver weight, hepatic triglycerides (TG), histological inflammatory scores, expression of inflammation-related and fibrosis-related genes, and hepatic retinoid levels. Compared to WT, KO mice on the HFD had hepatic enrichment of most cholesterol esters, monoglycerides, and certain sphingolipid species. Extended feeding with the WD for 10 months led to extensive liver injury, fibrosis, and hepatocellular carcinoma, with no difference between genotypes. Under alcohol exposure, KO and WT mice showed similar hepatic TG and liver enzyme levels. Interestingly, chow-fed KO mice showed significantly higher body and liver weights compared to WT mice, while KO mice on obesogenic diets had a shift toward larger lipid droplets.ConclusionsExtensive evaluation of Hsd17b13 deficiency in mice under several fatty liver-inducing dietary conditions did not reproduce the protective role of HSD17B13 loss-of-function mutants in human NAFLD. Moreover, mouse Hsd17b13 deficiency induces weight gain under RC. It is crucial to understand interspecies differences prior to leveraging HSD17B13 therapies.
| S-EPMC8627256 | biostudies-literature
Project description:The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex). A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain. Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities. The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity. Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component. This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions. The results support the view that His-602 is an active-site residue. The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent. Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids.
| S-EPMC1131597 | biostudies-other
Project description:Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. A single-nucleotide polymorphism (SNP), rs6834314, was associated with serum liver enzymes in the general population, presumably reflecting liver fat or injury. We studied rs6834314 and its nearest gene, 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13), to identify associations with histological features of NAFLD and to characterize the functional role of HSD17B13 in NAFLD pathogenesis. The minor allele of rs6834314 was significantly associated with increased steatosis but decreased inflammation, ballooning, Mallory-Denk bodies, and liver enzyme levels in 768 adult Caucasians with biopsy-proven NAFLD and with cirrhosis in the general population. We found two plausible causative variants in the HSD17B13 gene. rs72613567, a splice-site SNP in high linkage with rs6834314 (r2 = 0.94) generates splice variants and shows a similar pattern of association with NAFLD histology. Its minor allele generates simultaneous expression of exon 6-skipping and G-nucleotide insertion variants. Another SNP, rs62305723 (encoding a P260S mutation), is significantly associated with decreased ballooning and inflammation. Hepatic expression of HSD17B13 is 5.9-fold higher (P = 0.003) in patients with NAFLD. HSD17B13 is targeted to lipid droplets, requiring the conserved amino acid 22-28 sequence and amino acid 71-106 region. The protein has retinol dehydrogenase (RDH) activity, with enzymatic activity dependent on lipid droplet targeting and cofactor binding site. The exon 6 deletion, G insertion, and naturally occurring P260S mutation all confer loss of enzymatic activity. Conclusion: We demonstrate the association of variants in HSD17B13 with specific features of NAFLD histology and identify the enzyme as a lipid droplet-associated RDH; our data suggest that HSD17B13 plays a role in NAFLD through its enzymatic activity.
| S-EPMC6438737 | biostudies-literature